In recent years, an increased interest in the phyto-chemistry of the Annonaceae has been sparked by the bioactivity-directed isolation of the antileukemic Annonaceous acetogenin, uvaricin, from Uvaria acuminata. Acetogenins are C.sub.35 -C.sub.39 compounds and typically contain two long hydrocarbon chains, one of which connects a terminal 2,4-disubstituted-.gamma.-lactone to a variable number of tetrahydrofuran (THF) rings. The hydrocarbon chains contain a number of oxygenated moieties which can be hydroxyls, acetoxyls and/or ketones. Recently, single-ring acetogenins containing double bonds, epoxide compounds which lack THF rings and a compound lacking both epoxides and THF rings have been reported. These interesting newer compounds support the proposed polyketide origin of the Annonaceous acetogenins and provide additional clues to their biogenesis.
All acetogenins found to date contain multiple stereocentres, the elucidation of which often presents daunting stereochemical problems. Because of their waxy nature, the acetogenins do not produce crystals suitable for X-ray crystallographic analysis. Relative stereochemistries of ring junctions have typically been determined by comparison of natural compounds with synthetic model compounds and such methods have proven to be invaluable with the acetogenins. Recently, the absolute stereochemistries of the carbinol centers of acetogenins have been determined with the help of synthetic model compounds and high field nuclear magnetic resonance (NMR) analysis of their methoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters).
Most Annonaceous acetogenins are potently bioactive, but the mode of action of these compounds was unknown until Londerhausen et al. concluded in Pesticide Science, 33, 427-438 (1991), that they act to inhibit complex I of mitochondrial oxidative phosphorylation with an activity several times that of rotenone.
In accordance with the present invention there are provided novel, cytotoxic acetogenins and acetogenin derivatives. One group of acetogenins of this invention isolated from Asimina triloba are represented by the general formula EQU CH.sub.3 (CH.sub.2).sub.3 --R.sub.2 --R.sub.1
wherein R.sub.1, is a group of the formula ##STR1## and R.sub.2 is a divalent group selected from the group consisting of ##STR2## and acetylated derivatives thereof.
Another embodiment of this invention provides substantially pure compounds of the formula EQU CH.sub.3 (CH.sub.2).sub.11 --R.sub.3 --(CH.sub.2).sub.5 R.sub.1 '
wherein R.sub.1 ' is a group of the formula ##STR3## and R.sub.3 is selected from ##STR4## and acetylated derivatives thereof. The compound wherein --R.sub.3 -- is the divalent group (VI) is denominated goniocin, a naturally occurring acetogenin isolated from Goniothalamus giganteus. The compound wherein --R.sub.3 -- is the divalent group (VII) is prepared by epoxidation and subsequent acid-catalyzed cyclization of a previously reported acetogenin, gigantetronenin (also isolated from Goniothalamus giganteus), having the above formula wherein --R.sub.3 -- is a divalent group of the formula ##STR5##
Another aspect of this invention is directed to novel cyclic formaldehyde acetal derivatives of acetogenins having at least one 1,2-diol, 1,4-diol and/or 1,5-diol moiety in their structure. Such acetogenins include the art-recognized acetogenins bullatalicin, bullatanocin, squamocin, squamostatin A, gigantetrocin and goniothalamicin. The cyclic intramolecular formaldehyde acetal derivatives are prepared by reacting the acetogenin starting material with a 2-3 fold molar excess of chlorotrimethylsilane and dimethyl sulfoxide. Bis-cyclic formaldehyde acetals can be formed from acetogenins having two independent 1,2-, 1,4-, or 1,5-diol moieties. Preparation of the cyclic acetals significantly facilitates stereochemical structure elucidation. Moreover, the acetal derivatives show enhanced cytotoxicities against certain human tumor cell lines. The enhancement of the bioactive potencies and selectivities can permit improved utilization of many natural acetogenins.
The present invention further provides pharmaceutical formulations comprising an effective amount of an acetogenin compound for treating a patient having a tumor. As used herein, an effective amount of the acetogenin compound is defined as the amount of the compound which, upon administration to a patient, inhibits growth of tumor cells, kills malignant cells, reduces the volume or size of the tumors or eliminates the tumor entirely in the treated patient. Thus, the substantially pure compounds in accordance with this invention can be formulated into dosage forms using pharmaceutically acceptable carriers for oral or parenteral administration to patients in need of oncolytic therapy.
The effective amount to be administered to a patient is typically based on body surface area, patient weight, and patient condition. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, E. J., et al., Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area may be approximately determined from patient height and weight (see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., pages 537-538 (1970)). Preferred dose levels will also depend on the attending physicians' assessment of both the nature of the patient's particular cancerous condition and the overall physical condition of the patient. Effective anti-tumor doses of the present acetogenin compounds range from about 1 microgram per kilogram to about 200 micrograms per kilogram of patient body weight, more preferably between about 2 micrograms to about 100 micrograms per kilogram of patient body weight.
Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage and the possibility of co-usage with other therapeutic treatments including other anti-tumor agents, and radiation therapy.
The present pharmaceutical formulation may be administered via the parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in an isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carrier. In one preferred aspect of the present embodiment, the acetogenin compound is dissolved in a saline solution containing 5% of dimethyl sulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the present acetogenin compounds, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the acetogenin compounds. Alternatively, the present compounds can be chemically modified to enhance water solubility.
The present compounds can also be formulated into dosage forms for other routes of administration utilizing well-known methods. The pharmaceutical compositions can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal or a tablet. Capsules may comprise any well-known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with conventional procedure by compressing mixtures of the active acetogenins and solid carriers, and lubricants well-known to those familiar with the art. Examples of solid carriers include starch, sugar, bentonite. The compounds of the present invention can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder and a conventional fillers and tableting agents.